Inhibiting or downregulating glycogen synthase by creating premature stop codons using antisense oligonucleotides

ABSTRACT

The present disclosure relates to antisense oligonucleotides (AONs) for modulating the expression of glycogen synthase. AONs of the present disclosure may be useful in treating diseases associated with the modulation of the expression of the enzyme glycogen synthase, such as Pompe disease. Also provided by the present disclosure are compositions comprising AONs, as well as methods of down regulating mRNA coding for glycogen synthase, methods for reducing glycogen synthase in skeletal and cardiac muscle, and methods for treating Pompe disease.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority to U.S. ProvisionalPatent Application No. 62/002,294, filed May 23, 2014, which is herebyincorporated by reference in its entirety.

DESCRIPTION OF THE INVENTION

Field of the Invention

The present disclosure relates to antisense oligonucleotides (AONs) formodulating the expression of glycogen synthase. AONs of the presentdisclosure may be useful in treating diseases associated with themodulation of the expression of the enzyme glycogen synthase, such asPompe disease. Also provided by the present disclosure are compositionscomprising AONs, as well as methods of down regulating mRNA coding forglycogen synthase, methods for reducing glycogen synthase in skeletaland cardiac muscle, and methods for treating Pompe disease.

SUMMARY OF THE INVENTION

Pompe disease is an inherited disorder caused by the accumulation ofglycogen in the body's cells. This buildup of glycogen in the body,especially in skeletal and cardiac muscle, impacts the ability of thebody's organs and tissues to function normally.

There are three known types of Pompe disease, including classicinfantile-onset, non-classic infantile-onset, and late-onset. Theclassic form of infantile-onset begins within a few months of birth, andinfants with this disorder experience myopathy, hypotonia, hepatomegaly,and heart defects, and death from heart failure typically results withinthe first year of life if not treated. Non-classic infantile-onsetusually develops within the first year of life, and children with thisdisorder experience delayed motor skill development, progressive muscleweakness, and may have an enlarged heart. Serious breathing problems canoccur and children with this form of Pompe disease do not live pastearly childhood. The last type of Pompe disease, late-onset, may notbecome apparent until much later in a person's life, including even intoadulthood. Late-onset Pompe disease is usually milder than theinfantile-onset forms and typically does not involve the heart. However,people with this form of the disease experience progressive muscleweakness, which can lead to breathing problems and may eventually leadto respiratory failure.

Pompe disease is caused by mutations in the GAA gene. The GAA geneencodes for acid alpha-glucosidase, which is an enzyme that breaks downglycogen into the simple sugar glucose. Mutations in the GAA gene leadto the genetic deletion of acid alpha-glucosidase. As a result, glycogenbuilds up in the cells and leads to the symptoms associated with Pompedisease.

Pompe disease is currently treated by enzyme replacement therapy usingrecombinant GAA. However, this method of treatment is not alwaysentirely effective, and as a result, additional therapies for Pompedisease are needed.

Inhibiting the biosynthesis of glycogen is another potential means totreat patients with Pompe disease. So-called substrate reduction therapyis based on the inhibition of the main enzyme isoform responsible forbuilding the glycogen polymer in skeletal muscle, glycogen synthase 1.Three methods have been reported that accomplish glycogen synthase 1inhibition in Pompe mice: administration of a small interfering RNA(Douillard-Guilloux et al 2008); genetic knock down of the GSY1 gene inmice then crossed to Pompe mice (Douillard-Guilloux et al 2010); andinhibition of mTORC1 by administration of rapamycin (Ashe et al 2010).All three methods suppressed the accumulation of glycogen in Pompe mice.However, in the report by Ashe et al 2010 it was also reported that theadministration of rapamycin and recombinant human GAA was significantlymore effective at reducing glycogen accumulation in muscle tissue thaneither agent used alone. It was also revealed by Ashe et al thatglycogen synthase enzyme activity is greatly elevated in Pompe micesuggesting that the absence of GAA and accumulation glycogen interfereswith the normal regulation of the enzyme via phosphorylation. The doseof rapamycin needed to effect reduction of glycogen was the same as thatrequired for immunosuppression indicating that it could not be used as apharmacologic agent for the management of Pompe disease.

It is accordingly a primary object of the present disclosure to modulatethe expression of glycogen synthase, resulting in beneficial effects formammals who suffer from symptoms related to the buildup of glycogen inthe body's cells.

Glycogen synthase is an enzyme involved in converting glucose intoglycogen. In humans there are two different forms called isozymes orisoforms, glycogen synthase 1 and glycogen synthase 2, which are encodedfor by the genes GYS1 and GYS2, respectively. Glycogen synthase 1 isexpressed in muscles and other tissues, and glycogen synthase 2 isexpressed only in the liver. GYS1 encodes for a pre-mRNA that has 16exons. Exemplary human GYS1 sequences may be found at NCBI ReferenceSequence: NM_002103.4 also shown in SEQ ID NO.: 1 (DNA/RNA) and SEQ IDNO.: 2 (CDS) or via the Human Gene Nomenclature Committee at HGNC: 4706(see FIG. 1). Exemplary mouse GYS1 sequences may be found at NCBIReference Sequence: NM_030678.3 also shown in SEQ ID NO.: 3 (DNA/RNA)and SEQ ID NO.: 4 (CDS) or via the Human Gene Nomenclature Committee atMGI: 101805 (see FIG. 2).

According to the present disclosure, the modulation of pre-mRNA or mRNAtranscribed from GYS1 may result in the down-regulation of glycogensynthase protein and reduce glycogen synthase enzyme activity inskeletal and cardiac muscle, as well as treat and/or prevent thesymptoms associated with glycogen buildup in the muscles.

In one embodiment, the present disclosure relates to a method of downregulating mRNA coding for glycogen synthase comprising administering aneffective amount of an antisense oligonucleotide to an animal, whereinthe antisense oligonucleotide forms a sequence complementary to anucleic acid sequence encoding for glycogen synthase, and wherein thehybridization of the antisense oligonucleotide to the nucleic acidsequence encoding for glycogen synthase induces exon skipping. In oneembodiment, the present disclosure relates to a method of downregulating mRNA coding for glycogen synthase comprising administering aneffective amount of an antisense oligonucleotide to an animal, whereinthe antisense oligonucleotide forms a sequence complementary to anucleic acid sequence encoding for glycogen synthase, and wherein thehybridization of the antisense oligonucleotide to the nucleic acidsequence encoding for glycogen synthase induces translationalinhibition. In one embodiment, the present disclosure relates to amethod of down regulating mRNA coding for glycogen synthase comprisingadministering an effective amount of an antisense oligonucleotide to ananimal, wherein the antisense oligonucleotide forms a sequencecomplementary to a nucleic acid sequence encoding for glycogen synthase,and wherein the hybridization of the antisense oligonucleotide to thenucleic acid sequence encoding for glycogen synthase induces suppressionof polyadenylation.

One embodiment of the invention is method of down regulating mRNA codingfor glycogen synthase comprising administering an effective amount of anantisense oligonucleotide to an animal, wherein the antisenseoligonucleotide comprises a sequence complementary to a nucleic acidsequence encoding for glycogen synthase, and wherein the hybridizationof the antisense oligonucleotide to the nucleic acid sequence encodingfor glycogen synthase induces exon skipping. A further embodiment is ofthe invention is a method of down regulating mRNA coding for glycogensynthase comprising administering an effective amount of an antisenseoligonucleotide to an animal, wherein the antisense oligonucleotidecomprises a sequence complementary to a nucleic acid sequence encodingfor glycogen synthase, and wherein the hybridization of the antisenseoligonucleotide to the nucleic acid sequence encoding for glycogensynthase induces exon skipping, wherein the antisense oligonucleotide isa phosphorodiamidate morpholino oligo (also known as “PMO” or“morpholino”) or wherein the antisense oligonucleotide is a PMO linkedto a cell penetrating peptide (“CPP”).

A further embodiment is of the invention is a method of down regulatingmRNA coding for glycogen synthase comprising administering an effectiveamount of an antisense oligonucleotide to an animal, wherein theantisense oligonucleotide comprises a sequence complementary to anucleic acid sequence encoding for glycogen synthase, and wherein thehybridization of the antisense oligonucleotide to the nucleic acidsequence encoding for glycogen synthase induces exon skipping, whereinmRNA coding for glycogen synthase is reduced by up to 80%, up to 90% orup to 95%.

Additional objects and advantages of the present disclosure will be setforth in part in the description which follows, and in part willnaturally flow from the description, or may be learned by practice ofthe disclosed embodiments. The objects and advantages of the presentdisclosure will be realized and attained by means of the elements andcombinations particularly pointed out in the appended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate one (several) embodiment(s) ofthe invention and together with the description, serve to explain theprinciples of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the exemplary human GYS1 sequences as found at NCBI ReferenceSequence: NM_002103.4 also shown in SEQ ID NO.: 1 (DNA/RNA) and SEQ IDNO.: 2 (CDS).

FIG. 2 is the exemplary mouse GYS1 sequences as found at NCBI ReferenceSequence: NM_030678.3 also shown in SEQ ID NO.: 3 (DNA/RNA) and SEQ IDNO.: 4 (CDS).

FIG. 3: Gys1 mRNA levels are reduced in skeletal and cardiac muscles ofPompe mice treated with repeated intravenous injections of GS-PPMO. (A)Semi-quantitative PCR analysis was performed on pooled samples of RNAprepared from tissues of wild type and Pompe mice that received theindicated treatments to determine Gys1 transcript levels. Gys1 mRNAlevels (normalized to β-actin mRNA levels) were measured in the (B)quadriceps, (C) diaphragm, and (D) heart tissues. (E) Liver was examinedfor the impact of GS-PPMO on Gys2 mRNA levels. Data represent mean±SEM,n=4-5 mice per group. P<0.05, (*) compared to WT, (̂) compared tovehicle, (#) compared to rhGAA, ($) compared to GS-PPMO at 15 mg/kg.

FIG. 4: Gys1 protein levels are reduced in skeletal and cardiac musclesof Pompe mice treated with repeated intravenous injections of GS-PPMO.(A) Western blot analysis was carried out on pooled samples of proteinlysates to assess GYS1 protein levels in tissues of wild type and Pompemice that received the indicated treatments. GYS1 protein levels(normalized to gapdh protein levels), in the (B) quadriceps, (C)diaphragm, and (D) heart tissues were measured. (E) Liver was examinedfor the impact of GS-PPMO on Gys1/2 protein levels. Data representmean±SEM, n=4-5 mice per group. P<0.05, (*) compared to WT, (̂) comparedto vehicle, (#) compared to rhGAA, ($) compared to GS-PPMO at 15 mg/kg.

FIG. 5: Glycogen synthase activity is decreased in the quadriceps andheart muscles of Pompe mice treated with GS-PPMO. Glycogen synthaseactivity in the (A) quadriceps and (B) heart of wild type and Pompe micewas assayed as described in the Materials and Methods. Data representmean±SEM, n=4-5 mice per group. P<0.05, (*) compared to WT, (̂) comparedto vehicle, (#) compared to rhGAA, ($) compared to GS-PPMO at 15 mg/kg.

FIG. 6: Accumulation of lysosomal glycogen is abated in skeletal muscleof Pompe mice treated with GS-PPMO. Glycogen levels in the (A)quadriceps, (B) diaphragm, (C) heart and (D) liver of Pompe and wildtype mice were measured using the amplex red assay described in theMaterials and Methods. Data represent mean±SEM, n=4-5 mice per group.P<0.05, (*) compared to WT, (̂) compared to vehicle, (#) compared torhGAA, ($) compared to GS-PPMO at 15 mg/kg.

FIG. 7: Body weights of animals measured at the end of the study. Datais presented as mean±SEM, n=9-10 mice per group.

FIG. 8: Serum chemistries of Pompe mice treated with GS-PPMO compared tocontrol animals. (A-F) Levels of ALT, AST, LDH, CK, BUN, CrK (need tospell out all abbreviations) in serum collected 24 h after the finaldose. Data represent mean±SEM, n=9-10 mice per group. P<0.05, (*)compared to WT, (̂) compared to vehicle.

FIG. 9: Histopathological analysis of kidney and liver of Pompe and wildtype mice. Hematoxylin and eosin stained slides were prepared from micetreated with either vehicle of GS-PPMO as indicated. (A) Kidney sectionsof GS-PPMO treated Pompe mice show a normal architecture of proximalconvoluted tubules and glomeruli. (B) Livers of GS-PPMO-treated Pompemice exhibit the presence of Kupffer cells in hepatocytes.Magnification=40×.

Definitions

Glycogen synthase is an enzyme involved in converting glucose intoglycogen. In humans there are two different forms called isozymes orisoforms, glycogen synthase 1 and glycogen synthase 2, which are encodedfor by the genes GYS1 and GYS2, respectively. Glycogen synthase 1 islocated in muscles and other tissues, and glycogen synthase 2 is foundonly in the liver. GYS1 encodes for a pre-mRNA that has 16 exons.Exemplary human GYS1 sequences may be found at NCBI Reference Sequence:NM_002103.4 also shown in SEQ ID NO.: 1 (DNA/RNA) and SEQ ID NO.: 2(CDS) or via the Human Gene Nomenclature Committee at HGNC: 4706 (seeFIG. 1). Exemplary mouse GYS1 sequences may be found at NCBI ReferenceSequence: NM_030678.3 also shown in SEQ ID NO.: 3 (DNA/RNA) and SEQ IDNO.: 4 (CDS) or via the Human Gene Nomenclature Committee at MGI: 101805(see FIG. 2).

The term “RNA target” refers to an RNA transcript to which a morpholinobinds in a sequence specific manner. In some embodiments the RNA targetis one or more GSY1 mRNA or pre-mRNA molecules.

“Morpholino” or “morpholino antisense oligonucleotide” refer to anoligonucleotide analog composed of morpholino subunit structures, where(i) the structures are linked together by phosphorus-containinglinkages, one to three atoms long, preferably two atoms long, andpreferably uncharged or cationic, joining the morpholino nitrogen of onesubunit to a 5′ exocyclic carbon of an adjacent subunit, and (ii) eachmorpholino ring bears a purine or pyrimidine base-pairing moietyeffective to bind, by base specific hydrogen bonding, to a base in apolynucleotide. In some embodiments, the morpholino binds to an RNAtarget which blocks translation of the RNA target into a protein. Inother embodiments, the morpholino prevents aggregation of the RNA targetwith itself or with other cellular RNAs, proteins, or riboproteins, suchas, but not limited to, RNAs, proteins, and riboproteins associated withthe cellular mRNA splicing apparatus.

An “individual” can be a mammal, such as any common laboratory modelorganism, or any other mammal. Mammals include, but are not limited to,humans and non-human primates, farm animals, sport animals, pets, mice,rats, and other rodents.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention designed to alterthe natural course of the individual or cell being treated during thecourse of clinical pathology. Desirable effects of treatment include,but are not limited to, decreasing the rate of disease progression,amelioration or palliation of the disease state, and remission orimproved prognosis.

As used herein, “prevention” includes providing prophylaxis with respectto occurrence or recurrence of a disease or the symptoms associated witha disease in an individual. An individual may be predisposed to,susceptible to, or at risk of developing a disease, but has not yet beendiagnosed with the disease.

An “effective amount” or “therapeutically effective amount” refers to anamount of therapeutic compound, such as an antisense oligomer,administered to a mammalian subject, either as a single dose or as partof a series of doses, which is effective to produce a desiredtherapeutic effect. For an antisense oligomer, this effect is typicallybrought about by inhibiting translation or natural splice-processing ofa selected target sequence.

As used herein, the singular form “a”, “an”, and “the” includes pluralreferences unless indicated otherwise.

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

It is intended that every maximum numerical limitation given throughoutthis specification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

DETAILED DESCRIPTION OF THE INVENTION

Acid maltase or α-glucosidase (GAA) is a lysosomal enzyme that catalyzesthe breakdown of glycogen to glucose. Mutations in the GAA gene thatlead to a reduction in the amount or activity of the enzyme are themolecular basis of Pompe disease (glycogen storage disease type II).This autosomal recessive metabolic myopathy results as a consequence ofthe progressive accumulation of undegraded glycogen, primarily in thelysosomes of cardiac and skeletal muscle. Patients with Pompe disease(incidence of approximately 1 in 40,000) present with a broad spectrumof disease severity that is inversely correlated with the amount ofresidual enzyme activity. Complete loss of enzyme activity results in aninfantile presentation (so called “floppy babies”) with affectedindividuals rarely living beyond 2 years of age. Varying degrees ofresidual enzyme activity lead to a progressive myopathy in young adultsas well as older individuals that is invariably fatal.

Pompe disease is managed by periodic infusions of a recombinant enzyme(rhGAA) preparation that gained regulatory approval in 2006. Systemicinfusion of rhGAA has been shown in clinical trials to improvecardiomyopathy and prolong survival in children and to improve walkingability as well as stabilize pulmonary function in adults. However, itis evident from long-term survivors that despite the availability ofenzyme therapy, patients still present with some aspects of the disease.For example, residual muscle weakness and hearing loss are stillevident, and the risk for developing arrhythmias, dysphagia andosteopenia remain undiminished. These residual deficits may be due inpart to inefficient delivery of the enzyme to some of the affectedtissues or to the host immune response to the administered protein. Inresponse to these unmet medical needs, modified forms of rhGAAconjugated with mannose 6-phosphate-bearing oligosaccharides orengineered to express a portion of IGF-1 have been developed that showimproved delivery to muscle and bioactivity in animal studies. Inaddition, a small molecule chaperone that reportedly improves thestability of the enzyme and enhances glycogen clearance in Pompe mice isbeing tested clinically. Gene therapy with recombinant AAV vectorsencoding the enzyme deficient in Pompe disease is also being evaluatedas a treatment modality.

Substrate reduction that abates the production of glycogen representsyet another potential therapeutic strategy. The merits of this concepthave been demonstrated in the context of the lysosomal storage disordersGaucher and Fabry disease. Cytoplasmic glycogen polymers are synthesizedthrough the action of glycogen synthase, whose activity is suppressed byphosphorylation of serines 641 and 645 in a process controlled by themTORC1 pathway. Recent preclinical data have shown that glycogensynthase enzyme activity in Pompe mice is greatly elevated and that thisincreased activity could be suppressed by rapamycin treatment. TreatingPompe mice with rapamycin effectively reduced glycogen buildup inskeletal muscle, and when used in combination with rhGAA infusionslowered glycogen levels in skeletal muscle and diaphragm. Rapamycintreatment did not affect glycogen clearance in the heart, an organalready well served by rhGAA, perhaps due to the relatively high levelsof the cation-independent mannose 6-phosphate receptors in cardiactissue. An advantage of rapamycin as a substrate reduction approach wasthat its impact on glycogen synthase was restricted to muscle, with noeffect on the liver enzyme isoform. However, a disadvantage was that thelowest dose of rapamycin effective at reducing glycogen accumulation wasalso immunosuppressive.

The instant invention is directed to an alternative approach forreducing muscle glycogen levels in Pompe mice. Skeletal muscle glycogensynthase activity is the result of transcription of the Gys1 gene. Incontrast, liver synthase activity is generated mostly by expression ofthe Gys2 gene and its encoded enzyme produces glycogen as a ready storeof glucose for body-wide metabolism. Recent progress in developingtherapies for Duchene muscular dystrophy has demonstrated that it ispossible to deliver a therapeutically relevant dose of aphosphorodiamidate morpholino-based antisense oligonucleotide (PMO) forthe purpose of skipping mutant exons and generating a truncated, albeitfunctional dystrophin. The PMO dose needed to restore dystrophinsynthesis can be greatly reduced if it is conjugated to a cellpenetrating peptide (PPMO). Delivery of a therapeutically relevant doseof a phosphorodiamidate morpholino-based antisense oligonucleotide (PMO)for the purpose of skipping mutant exons was utilized to induce exonskipping of the Gys1 mRNA for the purpose of reducing its transcriptlevels, presumably and without being limited as to theory, via nonsensemediated decay, with concomitant reductions in the skeletal muscleenzyme. Treating Pompe mice with a PPMO targeting a specific sequence inexon 6 of Gys1 mRNA (GS-PPMO) reduces in a dose dependent manner theGys1 transcript in skeletal muscle and heart but not the Gys2 transcriptin liver. Likewise, the glycogen synthase protein level is reduced inskeletal muscle and heart and synthase activity is restored.Consequently, glycogen accumulation is completely abated in skeletalmuscle; the impact is less in the heart. These results indicate thatsubstrate reduction by antisense oligonucleotide (ASO)-mediated knockdown of skeletal muscle glycogen synthase is a therapy, or an adjuvanttherapy to enzyme replacement, for Pompe disease.

The present disclosure relates to oligomeric antisense compounds, i.e.,AONs, such as phosphorodiamidate morpholino (PMO) compounds, peptidenucleic acids (PNAs), 2′-O alkyl (e.g., methyl) antisenseoligonucleotides, and tricyclo-DNA antisense nucleotides for use inmodulating pre-mRNA and mRNA transcribed from GYS1. The presentdisclosure also includes other AONs that result from other nucleotidemodifications, such as for example, a modification to one or more of thenon-bridging oxygens in the phosphodiester linkage. Such modificationslead to, for example, phosphorothioates. The present disclosure relatesto any AON that specifically hybridize with one or more of pre-mRNA ormRNA transcribed from GYS1 and induce a reduction in glycogenaccumulation in a disease state. The AONs contemplated for use in theinstant invention include those attached to a cell-penetrating peptide(CPP) to enhance delivery. The AON-CPP may comprise multiple AON,including PMO AON, attached to a single CPP. In at least one embodimentof the present disclosure the multiple AON-CPP conjugate furthercomprises a cathepsin cleavable linker. The cathepsin cleavable linkercan occur in between the AON and the CPP or it can occur in a sequencesuch as AON-cathepsin linker-AON-cathepsin linker-CPP. In anotherembodiment, the multiple PMO-CPP conjugate further comprises a cathepsincleavable linker. The cathepsin cleavable linker can occur in betweenthe PMO and the CPP or it can occur in a sequence such as PMO-cathepsinlinker-PMO-cathepsin linker-CPP.

As used herein, an AON specifically hybridizes to a targetpolynucleotide, such as pre-mRNA or mRNA, when the AON hybridizes to thetarget under physiological conditions. In the context of the presentdisclosure, hybridization occurs via hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary purine and pyrimidine bases. For example, adenine (A) andthymine (T) are complementary nucleobases which pair through theformation of hydrogen bonds.

AONs, such as PMO compounds, of the present disclosure are complementaryto a target pre-mRNA or mRNA when hybridization occurs according togenerally accepted base-pairing rules, e.g., adenine (A)-thymine (T),cytosine (C)-guanine (G), adenine (A)-uracil (U). In particular,“complementary” as used herein refers to the capacity for precisepairing between two nucleobases. For example, if a base (B) at a certainposition of an AON is capable of hydrogen binding with a nucleotide atthe same position of a pre-mRNA or mRNA molecule, then the AON and thepre-mRNA or mRNA molecule are considered to be complementary to eachother at that position. The AON and pre-mRNA or mRNA target arecomplementary to each other when a sufficient number of correspondingpositions in each molecule are occupied by bases that can hydrogen bondwith each other. Thus, “specifically hybridizable” and “complementary”are terms which are used to indicate a sufficient degree ofcomplementarity or precise pairing such that stable and specific bindingoccurs between the AON, such as a PMO, and the pre-mRNA or mRNA target.Absolute complementarity, i.e., a 100% complementary base pair match, isnot necessary as long as the heteroduplex formed between the targetpre-mRNA or mRNA and the AON has sufficient stability to bring about thedesired effect such as a reduction in glycogen accumulation.

According to the present disclosure, an AON, such as a PMO, isspecifically hybridizable when binding of the AON to the target pre-mRNAor mRNA molecule interferes with the normal function of the targetpre-mRNA or mRNA molecule, and/or it brings about the desired effect,and there is a sufficient degree of complementarity to avoid intolerablenon-specific binding of the AON to a non-target sequence underconditions in which specific binding is desired, for example underphysiological conditions for in vivo applications or under conditions inwhich assays are performed for in vitro applications.

Such hybridization between an AON and pre-mRNA or mRNA interferes withtheir normal functions, such as translation of protein from the mRNA andsplicing of the pre-mRNA to yield one or more mRNA species. In at leastone embodiment of the present disclosure, the hybridization between theAON and pre-mRNA affects the splicing of the pre-mRNA to form stableRNA. In another embodiment the hybridization affects the translation ofglycogen synthase 1 from mRNA.

AONs according to the present disclosure include PMO compounds as wellas PNA compounds, phosphoramidate compounds, methylene methylimino(“MMI”) compounds, 2-O-methyl compounds and 2-methoxy ethyl compounds,wherein the oligonucleobase of each subunit are set forth in FIG. 1. Theoligonucleotide compounds are synthetic analogs of natural nucleicacids. In particular, instead of deoxyribose rings andphosphate-linkages, the oligonucleotide compounds comprise subunitscomprised of the respective oligonucleotide subunits shown below:

In the case of each of Formula 1-VI, B is a nucleotide base. The primarynucleobases are cytosine (DNA and RNA), guanine (DNA and RNA), adenine(DNA and RNA), thymine (DNA) and uracil (RNA), abbreviated as C, G, A,T, and U, respectively. A, G, C, and T appear in DNA, these moleculesare called DNA-bases; A, G, C, and U are called RNA-bases. Uracilreplaces thymine in RNA. These two bases are identical except thaturacil lacks the 5′ methyl group. Adenine and guanine belong to thedouble-ringed class of molecules called purines (abbreviated as R).Cytosine, thymine, and uracil are all pyrimidines (abbreviated as Y).

AON compositions can comprise morpholino oligonucleotide compositions.Morpholinos are synthetic molecules having a structure that closelyresembles a naturally occurring nucleic acid. These nucleic acids bindto complementary RNA sequences by standard nucleic acid base pairing.Structurally, morpholinos differ from DNA or RNA in that these moleculeshave nucleic acid bases bound to morpholine rings instead of deoxyriboseor ribose rings. Additionally, the backbone structure of morpholinosconsists of non-ionic or cationic linkage groups instead of phosphates.For example, replacement of anionic phosphates with the unchargedphosphorodiamidate groups eliminates ionization in the usualphysiological pH range, making morpholinos in organisms or cellsuncharged molecules. Morpholinos are most commonly used assingle-stranded oligos, though heteroduplexes of a morpholino strand anda complementary DNA strand may be used in combination with cationiccytosolic delivery reagents.

Unlike some other antisense structural types (e.g., phosphorothioates),morpholinos do not degrade their target RNA molecules. Instead,morpholinos act by “steric blocking,” i.e., binding to a target sequencewithin an RNA and sterically hindering molecules which might otherwiseinteract with the RNA. Bound to the 5′-untranslated region of messengerRNA (mRNA), morpholinos can interfere with progression of the ribosomalinitiation complex from the 5′ cap to the start codon. This preventstranslation of the coding region of the targeted transcript (called“knocking down” gene expression). Some morpholinos knock down expressionso effectively that after degradation of preexisting proteins thetargeted proteins become undetectable by Western blot.

Morpholinos can also interfere with pre-mRNA processing steps, usuallyby preventing splice-directing snRNP complexes from binding to theirtargets at the borders of introns on a strand of pre-RNA. Preventing U1(at the donor site) or U2/U5 (at the polypyrimidine moiety and acceptorsite) from binding can result in modified splicing, commonly leading tothe exclusion of exons from a mature mRNA transcript. Splicemodification can be conveniently assayed by reverse-transcriptasepolymerase chain reaction (RT-PCR) and is seen as a band shift after gelelectrophoresis of RT-PCR products.

Morpholinos have also been used to block intronic splice silencers andsplice enhancers. U2 and U12 snRNP functions have been inhibited bymorpholinos. Morpholinos targeted to “slippery” mRNA sequences withinprotein coding regions can induce translational frame shifts. Activitiesof morpholinos against this variety of targets suggest that morpholinoscan be used as a general-purpose tool for blocking interactions ofproteins or nucleic acids with mRNA.

In certain embodiments, the compositions of the present invention arecomposed of morpholino subunits linked together by unchargedphosphorus-containing linkages, one to three atoms long, joining themorpholino nitrogen of one subunit to the 5′ exocyclic carbon of anadjacent subunit, wherein the base attached to the morpholino group is apurine or pyrimidine base-pairing moiety effective to bind, bybase-specific hydrogen bonding, to a base in a polynucleotide. Thepurine or pyrimidine base-pairing moiety is typically adenine, cytosine,guanine, uracil or thymine. Preparation of such oligomers is describedin detail in U.S. Pat. No. 5,185,444, which is hereby incorporated byreference in its entirety. Variations can be made to this linkage aslong as they do not interfere with binding or activity. For example, theoxygen attached to phosphorus may be substituted with sulfur(thiophosphorodiamidate). The 5′ oxygen may be substituted with amino orlower alkyl substituted amino. The pendant nitrogen attached tophosphorus may be unsubstituted, monosubstituted, or disubstituted with(optionally substituted) lower alkyl. The purine or pyrimidine basepairing moiety is typically adenine, cytosine, guanine, uracil, thymineor inosine. The synthesis, structures, and binding characteristics ofmorpholinos are detailed in U.S. Pat. Nos. 5,698,685, 5,217,866,5,142,047, 5,034,506, 5,166, 315, 5,521,063, 5,506,337, andInternational Patent Application Publication No. WO 2008/036127 all ofwhich are incorporated herein by reference.

In some aspects, the morpholino antisense oligonucleotides of thepresent invention can be complementary to the pre mRNA sequences in thetranscript emanating from the GSY1 locus. In some embodiments, themorpholino antisense oligonucleotide is at least any of about 90%, 95%,or 100%, inclusive, including any percentages in between these values,identical to an mRNA target. In another embodiment, the morpholinoantisense oligonucleotide binds to the GSY1 mRNA transcript in asequence-specific manner. In some embodiments, the morpholino antisenseoligonucleotide comprises a 5′ amine modification. In anotherembodiment, the morpholino antisense oligonucleotide can be aphosphorodiamidate cationic peptide-linked morpholino antisenseoligonucleotide.

The morpholino antisense oligonucleotides described herein are linked toa cationic peptide which facilitates systemic delivery of the morpholinoantisense oligonucleotides into muscle cells. In general, a cationicpeptide as described herein can be 8 to 30 amino acid residues in lengthand consist of subsequences selected from the group consisting of RXR,RX, RB, and RBR; where R is arginine (which may include D-arginine), Bis β-alanine, and each X is independently —NH—(CHR¹)_(n)—C(O)—, where nis 4-6 and each R¹ is independently H or methyl, such that at most twoR¹'s are methyl. In some embodiments, each R¹ is hydrogen. In otherembodiments, the cationic peptide can be any of 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30amino acid residues in length. In another embodiment, the variable n is5, e.g. as in 6-aminohexanoic acid. In one embodiment, the cationicpeptide comprises the amino acid sequence Ac(RXRRBR)₂XB-, where Ac is anacetyl group. In another embodiment, the cationic peptide comprises theamino acid sequence Ac(RXR)₄XB-, where Ac is an acetyl group. Furtherinformation regarding synthesis and structure of cationiccell-penetrating peptides can be found in U.S. Patent ApplicationPublication No. 2009/0099066, the disclosure of which is incorporated byreference herein in its entirety.

In one aspect, the cationic peptide is linked directly to the morpholinoantisense oligonucleotide. In other embodiments, the cationic peptide islinked to the morpholino antisense oligonucleotide via a spacer moietylinked to the 5′ end of the morpholino antisense oligonucleotide. Thespacer moiety may be incorporated into the peptide during cationicpeptide synthesis. For example, where a spacer contains a free aminogroup and a second functional group (e.g., a carboxyl group or an aminogroup) that enables binding to another molecular moiety, the spacer maybe conjugated to a solid support used for peptide synthesis. Thereafter,the cationic peptide may be synthesized directly onto the spacer's freeamino group by standard solid phase techniques. In some embodiments, theAON-CPP compound may comprise multiple AON, including PMO AON, attachedto a single cationic peptide (CPP). In at least one embodiment of thepresent disclosure the multiple AON-CPP conjugate further comprises acathepsin cleavable linker. The cathepsin cleavable linker can occur inbetween the AON and the CPP or it can occur in a sequence such asAON-cathepsin linker-AON-cathepsin linker-CPP. In another embodiment,the multiple PMO-CPP conjugate further comprises a cathepsin cleavablelinker. The cathepsin cleavable linker can occur in between the PMO andthe CPP or it can occur in a sequence such as PMO-cathepsinlinker-PMO-cathepsin linker-CPP.

In another embodiment, the spacer moiety may be conjugated to thecationic peptide after peptide synthesis. Such conjugation may beachieved by methods well established in the art. In one embodiment, thelinker contains at least one functional group suitable for attachment tothe target functional group of the synthesized cationic peptide. Forexample, a spacer with a free amine group may be reacted with thecationic peptide's C-terminal carboxyl group. In some embodiments, thespacer moiety comprises:

In one embodiment, the cationic peptide-linked morpholino antisenseoligonucleotides have the following structure:

wherein R² is a cationic peptide (such as any of the cationic peptidesdisclosed herein), R³ is H, CH₃ or CH₂CONH₂, and R⁴ is a morpholinoantisense oligonucleotide comprising the sequence 5′-(AGC)_(n)-3′ (SEQID NO.: 5), 5′-(GCA)_(n)-3′ (SEQ ID NO.: 6), or 5′-(CAG)_(n)-3′ (SEQ IDNO.: 7), wherein n is any of about 5-25. In another embodiment, thecationic peptide-linked morpholino antisense oligonucleotides canfurther comprise 1 to 2 additional morpholino nucleotides on the 5′and/or 3′ end of the oligonucleotides.

In another aspect, the cationic peptide linked morpholino antisenseoligonucleotide comprises

wherein Ac is acetyl, R is arginine (which may include D-arginine), B isβ-alanine, each X is independently —NH—(CHR¹)_(n)—C(O)—, where n is 4-6and each R¹ is H, and R⁴ is a morpholino antisense oligonucleotidecomprising a therapeutic sequence.

In another aspect, the cationic peptide linked morpholino antisenseoligonucleotide comprises

wherein Ac is acetyl, R is arginine (which may include D-arginine), B isβ-alanine, each X is independently —NH—(CHR¹)_(n)—C(O)—, where n is 4-6and each R¹ is H, and R⁴ is a morpholino antisense oligonucleotidecomprising a therapeutic sequence.

In some embodiments, compound may include a variablesequence—spacer—linker according to any of the sequences of Table 2 orTable 3; wherein R is L-arginine or arginine; X is3-cis-aminocyclohexane or 1,3 cis-aminocyclohexane carboxylic acid; andZ is cis-2-aminocyclopentane-1-carbonyl or cis-(1R,2S)-2-aminocyclopentane carboxylic acid. In some embodiments, X can beany combination of 0, 1, or more residues that are R, X, and Z. In someembodiments, X can also include other types of residues, such asproline, glycine, or alanine, or additional modified or nonstandardamino acids. In some embodiments, the variable sequence includes alpha,beta, gamma, or delta amino acids, or cycloalkane structures. In someembodiments, the linker includes the sequence FS (SEQ ID NO.: 8). Insome embodiments, the linker includes the sequence FSQ (SEQ ID NO.: 9)or FSQK (SEQ ID NO.: 10), wherein F is phenyalanine, S is serine, K islysine and Q is glutamine. In some embodiments, the linker includes thesequence FxyB (SEQ ID NO.: 11), where x is any amino acid, standard ornonstandard, y is glutamic acid (E), aspartic acid (D), and lysine (K),serine (S), or threonine (T), and B is β-alanine or β-glycine.

TABLE 2 Hit Sequence Localization SEQ ID NO: 2C4Ac-RXXXXXRRR(Ahx)FSQG-OH Nucleus 12 4G9 Ac-RXXXXXXRR(Ahx)FSQG-OH Nucleus13 9F5 Ac-RXXXRXRXR(Ahx)FSQG-OH Nucleus 14 12G4 Ac-RRXXZXXXR(Ahx)FSQG-OHNucleus 15 12D10 Ac-RRRXXXXXR(Ahx)FSQG-OH Nucleus 16 12D11Ac-RXRXXXXXR(Ahx)FSQG-OH Nucleus 17 12E4 Ac-RRZXXXXXR(Ahx)FSQG-OHNucleus 18 21A5 Ac-RXXXXZXZR(Ahx)FSQG-OH Nucleus 19 11G1Ac-RXXZXRXXR(Ahx)FSQG-OH Cytosol 20 12D4 Ac-RRXRXXXXR(Ahx)FSQG-OHCytosol 21 13D2 Ac-RRZXXZXXR(Ahx)FSQG-OH Cytosol 22

In another embodiment, the cationic peptide linked to the morpholinoantisense oligonucleotide is one of the peptides in Table 3.

TABLE 3 Hit Sequence SEQ ID NO. 9H8 Ac-RXXXXXRXR(Ahx) 23 9H9Ac-RZXXXXRXR(Ahx) 24 9H11 Ac-RXZXXXRXR(Ahx) 25 1A2 Ac-RRRRRRRRR(Ahx) 2612D12 Ac-RZRXXXXXR(Ahx) 27 13D3 Ac-RXZXXZXXR(Ahx) 28 12D10Ac-RRRXXXXXR(Ahx) 29 2C4 Ac-RXXXXXRRR(Ahx) 30 4G9 Ac-RXXXXXXRR(Ahx) 3111F4 Ac-RXXXXRXXR(Ahx) 32 9F5 Ac-RXXXRXRXR(Ahx) 33 12D11Ac-RXRXXXXXR(Ahx) 34 20B7 Ac-RXXXXXXZR(Ahx) 35 20C4 Ac-RXXZXXXZR(Ahx) 365D4 Ac-RXXXRZXRR(Ahx) 37 9H7 Ac-RRXXXXRXR(Ahx) 38 5B1 Ac-RXXXZXXRR(Ahx)39 4H6 Ac-RXXZXXXRR(Ahx) 40 12D4 Ac-RRXRXXXXR(Ahx) 41 15A8Ac-RXXXXXZXR(Ahx) 42 12D8 Ac-RXZRXXXXR(Ahx) 43 12E3 Ac-RZXXXXXXR(Ahx) 4412H2 Ac-RXXZZXXXR(Ahx) 45 4G10 Ac-RZXXXXXRR(Ahx) 46 15H5Ac-RXXXXZZXR(Ahx) 47 11F1 Ac-RXRXXRXXR(Ahx) 48 21C7 Ac-RRXXZZXZR(Ahx) 4912E2 Ac-RXXXXXXXR(Ahx) 50 12E4 Ac-RRZXXXXXR(Ahx) 51 12G4Ac-RRXXZXXXR(Ahx) 52 21A5 Ac-RXXXXZXZR(Ahx) 53

When employed as pharmaceuticals, the antisense oligonucleotides,including cationic peptide-linked morpholino antisense oligonucleotides,disclosed herein can be formulated with a pharmaceutically acceptableexcipient or carriers to be formulated into a pharmaceuticalcomposition.

When employed as pharmaceuticals, the antisense oligonucleotides,including cationic peptide-linked morpholino antisense oligonucleotides,can be administered in the form of pharmaceutical compositions. Thesecompounds can be administered by a variety of routes including oral,rectal, transdermal, subcutaneous, intravenous, intramuscular, andintranasal. These compounds are effective as both injectable and oralcompositions. Such compositions are prepared in a manner well known inthe pharmaceutical art and comprise at least one active compound.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, one or more of the antisense oligonucleotides,including cationic peptide-linked morpholino antisense oligonucleotides,associated with one or more pharmaceutically acceptable excipients orcarriers. In making the compositions of this invention, the activeingredient is usually mixed with an excipient or carrier, diluted by anexcipient or carrier or enclosed within such an excipient or carrierwhich can be in the form of a capsule, sachet, paper or other container.When the excipient or carrier serves as a diluent, it can be a solid,semi-solid, or liquid material, which acts as a vehicle, carrier ormedium for the active ingredient. Thus, the compositions can be in theform of tablets, pills, powders, lozenges, sachets, cachets, elixirs,suspensions, emulsions, solutions, syrups, aerosols (as a solid or in aliquid medium), ointments containing, for example, up to 10% by weightof the active compound, soft and hard gelatin capsules, suppositories,sterile injectable solutions, and sterile packaged powders.

In preparing a formulation, it may be necessary to mill the activecompound to provide the appropriate particle size prior to combiningwith the other ingredients. If the active compound is substantiallyinsoluble, it ordinarily is milled to a particle size of less than 200mesh. If the active compound is substantially water soluble, theparticle size is normally adjusted by milling to provide a substantiallyuniform distribution in the formulation, e.g. about 40 mesh.

Some examples of suitable excipients or carriers include lactose,dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calciumphosphate, alginates, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterilewater, syrup, and methyl cellulose. The formulations can additionallyinclude: lubricating agents such as talc, magnesium stearate, andmineral oil; wetting agents; emulsifying and suspending agents;preserving agents such as methyl- and propylhydroxy-benzoates;sweetening agents; and flavoring agents. The compositions of theinvention can be formulated so as to provide quick, sustained or delayedrelease of the active ingredient after administration to the patient byemploying procedures known in the art.

The compositions are preferably formulated in a unit dosage form, eachdosage containing from about 5 mg to about 100 mg or more, such as anyof about 5 mg to about 10 mg, about 5 mg to about 20 mg, about 5 mg toabout 30 mg, about 5 mg to about 40 mg, about 5 mg to about 50 mg, about5 mg to about 60 mg, about 5 mg to about 70 mg, about 5 mg to about 80mg, or about 5 mg to about 90 mg, inclusive, including any range inbetween these values, of the active ingredient. The term “unit dosageforms” refers to physically discrete units suitable as unitary dosagesfor individuals, each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient or carrier.

The cationic peptide-linked morpholino antisense oligonucleotides areeffective over a wide dosage range and are generally administered in atherapeutically effective amount. It will be understood, however, thatthe amount of the cationic peptide-linked morpholino antisenseoligonucleotides actually administered will be determined by aphysician, in the light of the relevant circumstances, including thecondition to be treated, the chosen route of administration, the actualcompound administered, the age, weight, and response of the individualpatient, the severity of the patient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient/cationic peptide-linked morpholino antisense oligonucleotideis mixed with a pharmaceutical excipient or carrier to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules.

The tablets or pills of the present invention may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as corn oil,cottonseed oil, sesame oil, coconut oil, or peanut oil, as well aselixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. The compositions can be administered by the oral or nasalrespiratory route for local or systemic effect. Compositions inpharmaceutically acceptable solvents may be nebulized by use of inertgases. Nebulized solutions may be inhaled directly from the nebulizingdevice or the nebulizing device may be attached to a face mask tent, orintermittent positive pressure breathing machine. Solution, suspension,or powder compositions may also be administered, orally or nasally, fromdevices which deliver the formulation in an appropriate manner.

The overall effect of such interference with target pre-mRNA and mRNAtranscribed from GYS1 is selective modulation of the expression of GYS1and a change in glycogen accumulation. In the context of the presentdisclosure, “modulation” means either an increase (stimulation) or adecrease (inhibition) in the expression of a gene. According to thepresent disclosure, inhibition is the preferred form of modulation ofgene expression. The modulation of the expression of GYS1 is selectiveover the modulation of GYS2 according to the present disclosure becausethe pre-mRNA and RNA transcribed from GYS1 is targeted rather thanpre-mRNA and RNA transcribed from GYS2.

In at least one embodiment, the ASO compound has from 15-25 subunits ofa subunit selected from Formulas (I)-(VI). In another embodiment, theASO compound has from 20-25 subunits of a subunit selected from Formulas(I)-(VI). In yet another ASO, the ASO compound has about 25 subunits ofa subunit selected from Formulas (I)-(VI), such as from 24-26 subunits.

In a specific embodiment, the ASO, including a PMO, has a nucleobasesequence of one of the sequences of Table 4: Identification of activephosphorodiamidate morpholino oligomer (PMO) sequences designed totarget Gys1.

TABLE 4 % Gys1 mRNA SEQ ID Morpholino sequence remaining NO.  1TCAGGGTTGTGGACTCAATCATGCC  111 ± 14 54  2 AAGGACCAGGGTAAGACTAGGGACT99.7 ± 0.1 55  3 GTCCTGGACAAGGATTGCTGACCAT   81 ± 16 56  4CTGCTTCCTTGTCTACATTGAACTG  89  ± 7 57  5 ATACCCGGCCCAGGTACTTCCAATC  79 ± 10 58  6 CTGGACAAGGATTGCTGACCATAGT   72 ± 4 59  7AATTCATCCTCCCAGTCTTCCAATC   71 ± 20 60  8 TCCCACCGAGCAGGCCTTACTCTGA  83 ± 21 61  9 GACCACAGCTCAGACCCTACCTGGT  8.7 ± 1.5 62 10TCACTGTCTGGCTCACATACCCATA  7.8 ± 6.2 63

Gys1 mRNA levels were assessed in tibialis anterior muscles of C57Bl/6mice injected with individual PMOs as described in the Materials andMethods. The PMO sequence (in line 10) targeting exon 6 for skipping andreferred to herein as GS-PMO produced the greatest impact on Gys1 mRNAlevels as assessed by semi-quantitative PCR and was selected forsubsequent studies.

AONs, such as PMO compounds, according to the present disclosure thatspecifically hybridize to a target sequence at or near a splice site ofpre-mRNA transcribed from GYS1 can lead to inclusion of an intron in themRNA or to skipping both the intron and the exon near the splice sitetarget. Either event can lead to the introduction of a premature stopcodon, or a frame shift producing a nonsense mRNA. The inclusion of anexon usually leads to the inclusion of a stop codon in the reading frameof that intron. A frame shift caused by exon skipping also often leadsto a premature stop codon in the frame-shifted exon. Premature stopcodons are recognized and degraded by the nonsense-mediated machineryleading to exo and endo-nucleolytic mRNA degradation. (Bhuvanagiri etal., 2010) The degradation of mRNA transcribed from GYS1 may lead todown regulating mRNA coding for glycogen synthase 1, a reduced amount ofglycogen synthase 1, and ultimately alleviation of symptoms associatedwith the buildup of glycogen in the cells.

Accordingly, the present disclosure includes a method of down regulatingmRNA coding for glycogen synthase 1 comprising administering to ananimal an AON, such as a PMO, according to the present disclosure.

The present disclosure also includes a method for reducing glycogensynthase 1 in skeletal and cardiac muscle comprising administering to ananimal an AON, such as a PMO, according to the present disclosure.

EXAMPLES Methods and Materials

Design of Phosphorodiamidate Morpholino Oligomers

Phosphorodiamidate morpholino oligomers (PMOs) were designed tohybridize to Gys1 mRNA so as to invoke either exon skipping ortranslation inhibition as described by Morcos. The sequences designed toskip exons in Gys1 mRNA are as follows:

PMO 1 (5′-TCAGGGTTGTGGACTCAATCATGCC-3′) (SEQ ID NO.: 54) targeted theintronic sequence proximal to the splice acceptor site of intron 7;

PMO 2 (5′-AAGGACCAGGGTAAGACTAGGGACT-3′) (SEQ ID NO.: 55) targeted theintronic sequence proximal to the splice acceptor site of intron 4;

PMO 3 (5′-GTCCTGGACAAGGATTGCTGACCAT-3′) (SEQ ID NO.: 56) targeted theexon-intron boundary of exon 8;

PMO 4 (5′-CTGCTTCCTTGTCTACATTGAACTG-3′) (SEQ ID NO.: 57) targeted theintron-exon boundary of exon 5;

PMO 5 (5′-ATACCCGGCCCAGGTACTTCCAATC-3′) (SEQ ID NO.: 58) targeted theexon-intron boundary of exon 14;

PMO 6 (5′-CTGGACAAGGATTGCTGACCATAGT-3′) (SEQ ID NO.: 59), similar to PMO3 also targeted the exon-intron boundary of exon 8;

PMO 7 (5′-AATTCATCCTCCCAGTCTTCCAATC-3′) (SEQ ID NO.: 60) was designed toinhibit translation initiation by targeting a sequence 3′ to theinitiation codon of Gys1;

PMO 8 (5′-TCCCACCGAGCAGGCCTTACTCTGA-3′) (SEQ ID NO.: 61) targeted theexon-intron boundary of exon 7;

PMO 9 (5′-GACCACAGCTCAGACCCTACCTGGT-3′) (SEQ ID NO.: 62) targeted theexon-intron boundary of exon 5;

PMO 10 (5′-TCACTGTCTGGCTCACATACCCATA-3′) (SEQ ID NO.: 63) targeted theexon-intron boundary of exon 6.

Conjugation of Cell-Penetrating Peptides to Morpholino Oligonucleotides

PPMO conjugation was conducted as previously described by Abes et al. JControl Release. Dec. 1 2006; 116(3):304-313 with modifications. PeptideB (Ac(RXRRBR)2XB-OH) (SEQ ID NO.: 64) was activated in dimethylformamidecontaining O-(6-Chlorobenzotriazol-1-yl)-N,N,N,N″-tetramethyluroniumhexafluorophosphate (HCTU)/diisopropylethylamine (DIEA) at molar ratiosof 1:2 per moles of peptide at room temperature (RT). The Morpholino,GS1-ES6, (5′-TCACTGTCTGGCTCACATACCCATA-3′) (SEQ ID NO.: 63) with a 5′primary amine modification was dissolved in dimethylsulfoxide and addedto activated peptide at a 1.2-1.5:1 molar ratio of peptide:ASO andallowed to react at RT for 2 h; when completed the reaction was quenchedwith water. PPMO conjugates were separated from unbound PMO by isolationover carboxymethyl sepharose and eluted in 2M guanidine-HCl, 1M NaCl, pH7.5, 20% acetonitrile. The eluate was dialyzed against several bufferexchanges of 0.1mM NaHCO3 in a dialysis cassette with molecular weightcut-off of 3,000 Da. The dialyzed PPMO was quantified byspectrophotometric absorbance in 0.1N HCl at 265 nm, frozen, andlyophilized. Molecular weight of conjugated GS1-ES6 PPMO was confirmedby MALDI mass spectrometry.

In-Vivo Experiments

Animal experiments were conducted in accordance with the Guide for theCare and Use of Laboratory Animals (US Department of Health and HumanServices, NIH Publication No. 86-23) and by Genzyme's IACUC committee.

Intramuscular TA Injections

Six week-old C57BL/6 mice were anesthetized with isoflurane and thetibialis anterior (TA) muscle was injected as previously described[Wheeler T M et al., J Clin Invest. December 2007; 117(12):3952-3957].TA muscles were injected with 12 uL of 0.4 U/μL bovine hyaluronidase 2hours before PMO injection and electroporation. One TA was injected with20 μg (1 μg/μl) of various PMOs (Table 4) and the contralateral TA with20 μI phosphate buffered saline (PBS). Immediately following injection,the muscle was electroporated using the parameters of 100 V/cm, 10pulses at 1 Hz, and 20 ms duration per pulse. Mice were euthanized twoweeks after electroporation and TA muscle collected and snap frozenuntil analysis.

Systemic Administration

Six week-old male and female GAA^(−/−) and C57BL/6 mice were employed toevaluate the efficacy of substrate inhibition using peptide-linkedmorpholinos. Tissues were collected from a cohort of animals at thestart of the studies to serve as a baseline reference for the glycogenaccumulation assay (n=10). GS-PPMO was dissolved in PBS and administeredat 15 or 30 mg/kg bodyweight by tail vein injection once every 2 weeksfor a total of 12 weeks (n=9-10). The positive control, rhGAA, wasreconstituted in a buffer consisting of 25 mM sodium phosphate pH 6.2,2% mannitol, and 0.005% polysorbate 80 and 20 mg/kg administered by tailvein injection once every 2 weeks for 12 weeks (buffer, n=10; rhGAA,n=10). To minimize the potential for a hypersensitivity reaction torhGAA, mice were intraperitoneally pretreated with 5 mg/kgdiphenhydramine starting at the third dose of rhGAA. Two weeks after thefinal dose mice were euthanized, tissues collected and snap frozen inliquid nitrogen for in vitro analyses or fixed in 10% neutral bufferedformalin for histological analysis.

In-Vitro Experiments

RNA Analysis

Total RNA was isolated from frozen tissue using a commercially availablekit with optional DNA digestion. RT-PCR was conducted using customprimers used for cDNA synthesis and PCR amplification. Primer sequences:Gys1 forward, 5′-CTGGCGCTGTGGACTTCTA-3′ (SEQ ID NO.: 65), Gys1 reverse,5′-ACACTGGTGGGAAAGACTGC-3′ (SEQ ID NO.: 66), Gys2 forward,5′-CCAGCTTGACAAGTTCGACA-3′ (SEQ ID NO.: 67), Gys2 reverse,5′-AAACACCCCAAGGTGACAAC-3′ (SEQ ID NO.: 68), b-actin forward,5′-AGCCATGTACGTAGCCATCC-3′ (SEQ ID NO.: 69) and b-actin reverse,5′-CTCTCAGCTGTGGTGGTGAA-3′ (SEQ ID NO.: 70). RT-PCR products (25 cycles)were separated on 2% agarose gels containing ethidium bromide andscanned on a bio-imaging system. Band intensity was quantified usingImage J software. Levels of Gys1 and Gys2 mRNA was determined relativeto beta actin.

Preparation of Tissue Homogenates

Tubes containing frozen tissues with 6x (vol/wt) homogenization bufferdesigned to inhibit phosphatases and proteases (20 mM Tris/HCL, pH 7.5,150 mM NaCl, 25 mM B-glycerophosphate, 20 mM Sodium Fluoride, 1 mMSodium Orthovanadate, 2 mM Sodium Pyrophosphate, 2 mM EDTA and completeprotease inhibitor cocktail were homogenized. Lysates were frozen for 24hours at −80 C. Thawed lysates were centrifuged 16.1 rcf for 15 minutesat 4 C., the supernatants were aliquoted and stored at −80 C. Proteindetermination of the lysates was performed with a Micro BCA kit.

Western Blot Analysis of Tissue Lysate

50-100 μg of tissue homogenate was boiled in 2× sample buffer containingdithiothreitol. The lysate was then applied to a 4-15% precastTris/HCl-polyacrylamide gel. Proteins were transferred to nitrocellulosewith a dry blot apparatus. The blots were blocked overnight with 3% milkand the appropriate antibody added at a final concentration of 0.02-0.08ng/ml and incubated for 1 hr at room temp. The blot was then incubatedwith an HRP-conjugated secondary antibody for 1 hr at room temperatureand treated with an ECL substrate detection kit as described by themanufacturer. Protein band intensity was quantified using Image Jsoftware. Levels of glycogen synthase 1 and 2 protein was determinedrelative to GAPDH.

Glycogen Synthase Activity Assay

Glycogen synthase activity in tissue lysates was measured using a gelfiltration radioactivity assay as described previously [Niederwanger Aet al., J of Chromatography B, 2005; 820:143-145]. A 60 μL reactionsolution consisting of 10 μg of protein lysate (2 ng/μL), 4% glycogen,30 mM UDP-glucose, 4.5 mM glucose-6-phosphate, homogenization buffer(described above) and labeled uridine diphosphate glucose[Glucose-¹⁴C-U] was incubated in a 37° C. water bath for 1 h, thereaction was stopped with 0.6N perchloric acid, and 50 μL of thereaction was loaded onto a quick spin (G-50) sephadex columns andcentrifuged at 1000×g for 4 min. The eluted radiation was added to LSCcocktail and radiation was measured using a scintillation counter.Enzyme activity was calculated by determining the amount ofUDP-[U-¹⁴C]-glucose incorporated into glycogen per minute per milligramof protein

Measurement of Tissue Glycogen

Tissue glycogen levels were determined as previously described [ZieglerR J et al., Hum Gene Ther. June 2008; 19(6):609-21]. Fluorescence wasdetected and analyzed using a micro-plate reader, 530 nm excitation and590 nm emission, with acquisition and analysis software. Rabbit liverglycogen was used to construct the standard curve. Glycogen levels weredetermined by subtracting the glucose levels in the undigested samplesfrom those in the digested samples.

Serum Chemistry

Whole blood was collected in serum separator tubes from theretro-orbital plexus of anesthetized mice one hour after the final doseof GS-PPMO. Blood was allowed to clot for thirty minutes and thencentrifuged at 1300×g for fifteen minutes. Serum was dispensed asaliquots and frozen at −20° C. until analysis. In-vitro diagnosticquantitative determination of chemistry analytes in serum was performedby spectrophotometric methods at 37° C.

Histology

Kidney and liver were collected from mice following euthanasia, fixedfor up to 72 h in 10% neutral buffered formalin and processed forparaffin embedding. Serial 5 μm-thick sections were generated andstained with hemotoxylin and eosin solution. A board certifiedveterinary pathologist, blinded-to the treatments, evaluated the slidesfor qualitative analysis.

Statistical Analysis

Data is expressed as mean±SEM. Data analysis was performed using one wayANOVA and Newman-Keuls post-hoc. A probability value of P<0.05 wasconsidered to be statistically significant.

PMO-based antisense oligonucleotide confers selective knockdown of Gys1mRNA in murine muscle.

A collection of PMO antisense oligonucleotides was designed toselectively reduce the expression of the isoform of glycogen synthasefound mainly in skeletal muscle and heart with the potential to induceexon skipping in the cognate Gys1 but not the Gys2 transcript. Exonskipping was designed to introduce a premature stop codon into the Gys1transcript to effect the production of an unstable mRNA prone tononsense-mediated decay. Incorporation of a nonsense codon would also beexpected, after translation, to lead to a non-functional enzyme.

Candidate ASOs were first tested by direct injection into the TA muscleof mice followed by electroporation. One week later Gys1 mRNA levelswere quantified. Twelve ASOs were tested and two resulted in whatappeared to be a substantial reduction in Gys1 mRNA (Table 4). One PMOsequence in particular (number 10 in Table 4) targeted the skipping ofexon 6 and was evaluated further.

The selected PMO was synthesized with a 5′ primary amine to facilitateconjugation of a well-characterized arginine-rich cell penetratingsequence that had been shown previously to facilitate muscle delivery.This conjugated PMO (GS-PPMO) was injected through a tail vein intoPompe mice once every two weeks for a total of 12 weeks. Age-matchedPompe mice administered saline vehicle or 20 mg/kg recombinantα-glucosidase (rhGAA) on the same schedule served as treatment controls.Age-matched wild type (57Bl/6) mice served as untreated controls.Analysis of tissue extracts from Pompe mice at the end of the studyshowed that treatment with either 15 or 30 mg/kg GS-PPMO significantlyreduced Gys1 mRNA levels in the quadriceps and diaphragm muscle (FIGS.3A, 3B and 3C). Gys1 mRNA levels were also dramatically reduced in theheart, but only after treatment with the higher dose (FIG. 3D). Nosignificant changes were seen in the steady state levels of Gys2 mRNA inthe liver, indicating that GS-PPMO-mediated knockdown was specific forthe muscle isoform of glycogen synthase (FIG. 3E). As expected, treatinganimals with rhGAA had no impact on Gys1 mRNA levels in skeletal muscleor heart or the Gys2 mRNA levels in liver.

Systemic administration of GS-PPMO to Pompe mice selectively reducesglycogen synthase 1 levels.

Western blots were used to assess the degree to which the reductions inGys1 mRNA led to concomitant reductions in glycogen synthase protein inmuscle and liver. In Pompe mice, glycogen synthase protein was found tobe substantially elevated in the skeletal muscle and in the heartcompared to C57Bl/6 control animals (FIG. 4 A-E). This increase was notdue to higher levels of mRNA (FIG. 3) and may therefore be related to anincrease in the stability of the enzyme in these Pompe tissues. However,there were no differences in the levels of glycogen synthase in theliver of Pompe mice compared to those of wild type control animals.

Treatment of Pompe mice with GS-PPMO (15 or 30 mg/kg) lowered the amountof glycogen synthase in the quadriceps and diaphragm to wild type levels(FIGS. 4A, 4B and 4C). Treatment also reduced the elevated levels ofglycogen synthase in the Pompe mouse heart, and achieved completecorrection at the higher (30 mg/kg) dose (FIG. 4D). Neither dose ofGS-PPMO affected the amount of total glycogen synthase in the liver(FIG. 4E). Treating Pompe mice with rhGAA also did not significantlyalter the amount of glycogen synthase in the tissues tested.

Systemic administration of GS-PPMO reduces glycogen synthase activity inthe skeletal muscle and heart of Pompe mice.

Treating Pompe mice with GS-PPMO led to complete correction of theelevated glycogen synthase activity in the quadriceps (FIG. 5A). Areduction of enzyme activity in the heart was observed only in Pompemice treated at the higher dose of 30 mg/kg GS-PPMO (FIG. 5B) whereas anapparent increase in activity was noted at the lower dose. In general,these findings are consistent with the mRNA and protein measurementsnoted above. Treating Pompe mice with rhGAA (at the dose tested) had noeffect on glycogen synthase activity in the skeletal muscle but loweredthat in the heart to normal levels. This differential response totreatment with the recombinant enzyme is consistent with previousreports in Pompe mice.

Systemic treatment with GS-PPMO abates accumulation of tissue glycogenin Pompe mice.

Tissue extracts were subjected to quantitative glycogen analysis todetermine whether the noted reductions in glycogen synthase proteinlevels and activity in GS-PPMO-treated Pompe mice also resulted in aconcomitant lowering of lysosomal glycogen accumulation. Treating Pompemice with GS-PPMO led to a dose dependent decrease in glycogenaccumulation in the quadriceps and heart (FIGS. 6A and 6C), and areduction to the level found in normal control mice in the diaphragm atboth doses tested (FIG. 6B). In the quadriceps and diaphragm of Pompemice treated with 30 mg/kg GS-PPMO, the levels of glycogen were reducedto those found in wild type C57Bl/6 mice. As expected, Pompe micetreated with 20 mg/kg rhGAA showed a partial reduction in glycogenlevels in the quadriceps, diaphragm and a greater reduction in the heart(FIG. 6). Neither treatment with rhGAA nor GS-PPMO (15 mg/kg) had animpact on glycogen levels in the liver. However, treatment with thehigher dose (30 mg/kg) of GS-PPMO did result in a partial reduction inliver glycogen levels (FIG. 6D).

Systemic administration of GS-PPMO does not elicit overt changes inhistopathology and blood chemistry.

The potential toxicologic impact of GS-PPMO treatment and knockdown ofGYS1 mRNA was evaluated to assess the therapeutic index associated withadministering a PMO-based ASO for Pompe disease. Pompe mice treated witheither dose of GS-PPMO did not demonstrate significant differences inweight gain from mice in the control cohort (FIG. 7). Examination ofblood biomarkers of liver, muscle and kidney damage also did not revealany deviations from those noted in control mice (FIG. 8). Finally,histological analysis of the kidney and liver of GS-PPMO-treated Pompemice revealed normal architecture and the absence of discernible lesions(FIG. 9). These data suggests that systemic administration of GS-PPMO iswell tolerated, at the doses tested.

GS-PPMO was capable of provoking Gys1 mRNA decreases in quadriceps,diaphragm and heart in a dose dependent manner. The bioactivity seen inthe heart was only significant at the higher dose tested, a findingconsistent with PPMOs tested for exon skipping of dystrophin (data notshown). GS-PPMO activity at the mRNA level appeared to be sequencespecific as there was no impact on the liver isoform, Gys2. This findingwas expected given that GS-PPMO is complementary to intron sequence inGys1. The fact that GS-PPMO appears specific for the muscle enzymesuggests that its action will not interfere with systemic glucosemobilization in Pompe patients, which is governed by the predominantliver enzyme encoded by the Gys2 gene. Also as expected, administrationof rhGAA had no impact on the steady state level of Gys1 or Gys2 mRNA inany tissue tested.

The GS-PPMO mediated knock down of Gys1 mRNA greatly reduced the amountof Gys1 protein in quadriceps and diaphragm at both doses tested, andalso in the heart at the higher dose. These findings are impressivegiven the elevation of the protein in these tissues seen in Pompe micecompared to control animals. There was no change in the level of liverGys2 protein which is consistent with the designed specificity ofGS-PPMO towards Gys1 mRNA. The effect of GS-PPMO treatment on glycogensynthase enzyme activity in the quadriceps and heart was furtherevaluated and substantial reduction was found in both tissues as well assome noteworthy differences. Glycogen synthase activity was considerablyelevated in the quadriceps and heart of Pompe mice; a finding consistentwith the protein levels cited above and previous reports. Treating Pompemice with GS-PPMO reduced activity in these tissues to very near thewild type levels found in C57Bl/6 mice at both doses tested. This wasalso true in the heart but only with the higher dose employed. It isremarkable to note that even with the elevated level of GS-activity inthe untreated Pompe heart, the administration of GS-PPMO (15 mg/kg)increased GS-activity further by 200-fold. Glycogen synthase activity isregulated at the level of protein phosphorylation that is controlled byenvironmental conditions through the mTOR pathway.

The effect of rhGAA and GS-PPMO treatment on glycogen build up in Pompemice was also assessed. Analysis of quadriceps and diaphragm aftertreatment with rhGAA at 20 mg/kg both revealed modest declines inglycogen compared to vehicle-treated Pompe mice. Treatment with GS-PPMOwas significantly more effective at abating glycogen build up in thequadriceps and diaphragm, the latter tissue being equally amenable totreatment with either dose. The heart showed complete abatement ofglycogen build up by rhGAA treatment to a level even below that found inuntreated C57Bl/6 control mice. This is in contrast to theaforementioned increase in GS activity and may be due to the well-notedpresence of CI-M6P receptors in the heart permitting greater efficacy ofrhGAA in that tissue. GS-PPMO treatment resulted in a modestdose-dependent decline of glycogen build up in heart.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

What is claimed is:
 1. A method of down regulating mRNA coding forglycogen synthase comprising administering an effective amount of anantisense oligonucleotide to an animal, wherein the antisenseoligonucleotide comprises a sequence complementary to a nucleic acidsequence encoding for glycogen synthase, and wherein the hybridizationof the antisense oligonucleotide to the nucleic acid sequence encodingfor glycogen synthase induces exon skipping.
 2. The method of claim 1,wherein the antisense oligonucleotide is a PMO.
 3. The method of claim1, wherein the antisense oligonucleotide is a PMO linked to a CPP. 4.The method of claim 1, wherein the antisense oligonucleotide is selectedfrom an oligonucleotide comprising subunits of one of Formula I-VI. 5.The method of claim 1, wherein mRNA coding for glycogen synthase isreduced by 80%.
 6. The method of claim 1, wherein mRNA coding forglycogen synthase is reduced by 90%.
 7. The method of claim 1, whereinmRNA coding for glycogen synthase is reduced by 95%.
 8. The method ofclaim 1, wherein the effective amount ranges from 5 to 500 mg per dose.9. The method of claim 1, wherein the compound is administeredintravenously.
 10. The method of claim 1, wherein the down regulation ofmRNA coding for glycogen synthase occurs in skeletal and cardiac muscle.11. The method of claim 1, wherein the antisense oligonucleotide is atleast 85% complementary to the nucleic acid sequence encoding forglycogen synthase.
 12. The method of claim 1, wherein the antisenseoligonucleotide is at least 90% complementary to the nucleic acidsequence encoding for glycogen synthase.
 13. The method of claim 1,wherein the antisense oligonucleotide is at least 95% complementary tothe nucleic acid sequence encoding for glycogen synthase.
 14. A methodfor reducing glycogen synthase in skeletal and cardiac muscle comprisingadministering to an animal an effective amount of an antisenseoligonucleotide to an animal, wherein the antisense oligonucleotidecomprises a sequence complimentary to a nucleic acid sequence encodingfor glycogen synthase, and wherein the hybridization of the antisenseoligonucleotide to the nucleic acid sequence encoding for glycogensynthase induces exon skipping.
 15. A method for treating Pompe diseasecomprising administering to an animal an effective amount of anantisense oligonucleotide to an animal, wherein the antisenseoligonucleotide comprises a sequence complimentary to a nucleic acidsequence encoding for glycogen synthase, and wherein the hybridizationof the antisense oligonucleotide to the nucleic acid sequence encodingfor glycogen synthase induces exon skipping.